Regulation of the catalytic unit (C) of adenylate cyclase through interaction with both the G/F component and by cytosolic agents is important in understanding the mechanism by which hormones, cholera toxin, calmodulin, other agents stimulate cellular cAMP production. To study that regulation, we are attempting to isolate and characterize C from various tissues. The requirements for stability and activity of C from bovine brain were investigated. C, separated from G/F by gel filtration of a CHAPS-solubilized particulate fraction, is inactivated by incubation at 30 C; ATP provided almost complete stabilization. Whereas catalytic activity in these preparations is virtually completely dependent on the presence of Mg-++ or Mn-++, stabilization by ATP does not require divalent cations. Calcium plus calmodulin and phosphatidylcholine plus lysophosphatidylcholine also stabilize C. In addition, these agents activate C. These findiang, in addition to defineing some properties of C that are apparently independent of G/F, may well find practical application in procedures for purification and/or further characterization of C.